The major causes of mortality and morbidity in industrialized societies result from the multiple downstream sequelae of high fat diet-induced obesity (HFDIO). Previously, we demonstrated the importance of calciumindependent phospholipase A2?(iPLA2?) as a central enzymic mediator integrating cellular signaling and organismal bioenergetics through the generation and characterization of a global iPLA2??KO mouse. Remarkably, the iPLA2??knockout mouse was completely resistant to HFDIO and the development of insulin resistance. However, due to the multiple organ systems affected in the whole animal iPLA2??knockout, the mechanistic roles of iPLA2??in each tissue contributing to the complete resistance of this mouse to HFDIO are unknown. The overarching goal of the proposed research is the mechanistic determination of the tissue- and Organelle-specific roles of iPLA2??in promoting inflammation, maladaptive cellular signaling and dysfunctional bioenergetics that result in insulin resistance and the pathologic end-organ sequelae of HFDIO. Accordingly, we generated mice containing a floxed construct of the iPLA2??active site which has been crossed with tissue specific Cre mice resulting in the generation of hepatocyte-specific and skeletal muscle myocyte-specific iPLA2??knockout mice. The proposed research will synergistically use these enabling genetic models in conjunction with the integrated lipidomics and metabolomics platforms we developed to identify the mechanisms through which iPLA2??participates in the development of obesity, inflammation, and insulin resistance during HFDIO. The first specific aim will identify the roles of hepatocyte-specific iPLA2??in mediating alterations in the generation of lipid 2nd messengers, lipid metabolism, hepatosteatosis and bioenergetics during HFDIO unencumbered by the effects of iPLA2??loss of function in other cell types that are present in the germline knockout. In Specific Aim 2, we will examine the effects of skeletal muscle myocyte-specific ablation of iPLA2??on skeletal muscle metabolism, insulin resistance and mitochondrial dysfunction that are present during high fat feeding in WT mice, but are rescued in the germline knockout mouse. Finally, in Specific Aim 3, we will determine the interactive mechanistic roles of the organelle-specific isoforms of iPLA2??through the transgenic reintroduction of either the mitochondrial-specific or peroxisomal-specific isoforms of iPLA2??into the hepatocyte-specific iPLA2??knockout mouse. Moreover, the mechanisms through which lipid 2nd messengers generated by iPLA2??in hepatocytes, skeletal muscle myocytes and adipocytes mediate inter-organ communication between these metabolically interwoven tissues will be identified through synergistic highly penetrating technologies we have developed/refined. Through this multidisciplinary approach employing novel genetic reagents, high mass accuracy mass spectrometry technologies and integrated chemophysiologic approaches, novel pharmacologic targets to attenuate the sequelae of HFDIO can be identified.